Overview: This research program addresses basic molecular and physiological processes of nociceptive (pain-sensing) transmission in the peripheral and central nervous systems (CNS) and new ways to effectively control pain. The molecular research is performed using animal and in vitro cell-based models. We concentrate on primary afferent pain-sensing neurons located in dorsal root ganglion (DRG) that send nerve fibers to skin and deep tissues and make connections within dorsal spinal cord, which is the first CNS site of synaptic information processing for pain. The mechanisms of transduction of physical pain stimuli are investigated through models of pathophysiological damage or using reductionist preparations such as primary DRG cultures or heterologous expression systems of ion channels or receptors. Our goals are (a) to understand the molecular and cell biological mechanisms of acute and chronic pain at the initial steps in the pain pathway, (b) to investigate mechanisms underlying human chronic pain disorders, (c) to explore neuronal plasticity and altered gene expression in persistent pain states, and (d) to use this knowledge to devise new treatments for pain. New Treatments for Pain: We address the new treatment goal through translational research coupled with human clinical trials to develop and introduce new molecular interventions for severe pain. Studies with the TRPV1 agonist resiniferatoxin (RTX) have resulted in a Phase I clinical trial for in patients with intractable pain from advanced cancer. RTX activates an influx of sodium and calcium ions into nerve endings and once bound to TRPV1, RTX props open the ion channel causing an intracellular calcium cytotoxicity. Depending on the route of administration this disables TRPV1 pain-sensing nerve endings or axons (i.e., the nerve fiber) or deletes the neuron entirely. RTX produces very effective pain control in pre-clinical models. The central route involves administration into the cerebrospinal fluid around the spinal cord (intrathecal). We have treated 13 patients with pain from advanced cancer. This study is nearly complete. We also published a study of injections around or directly into sensory ganglia, and, based on this approach, we will commence a new protocol for more localized cancer pain problems such as osteosarcoma. Peripheral routes of RTX administration include direct injection into skin, joints, nerve bundles, or topically. Analgesia by these routes is long-lasting, but temporary, since nerve endings and axons regrow. Peripheral administration formed the basis of three reports, one in which we successfully treated experimental burn pain, another in which we successfully used RTX as a preemptive analgesic for surgical incision pain, and a third in which we successfully treated clinical osteoarthritis (OA) pain by intraarticular injection in client owned dogs. The canine results demonstrated both efficacy and a long duration of action (4 to >12 months) and strongly reinforce translation to human patients. Early Translational Investigations: In collaboration, we have also examined the pharmacological activity of polyunsaturated ethanolamines and linoleic acid metabolites. These putative endovanilloids. could function as endogenous orthosteric agonists at TRPV1. In this cycle, we evaluated tissue biosynthetic pathways for new endogenous lipids. We published our discovery of two previously unknown lipids. One sensitized nerves to nociceptive stimuli, the other caused itch and, in humans, were associated with headache and itch conditions, respectively. In ongoing allied studies, we examined small chemicals that act as positive allosteric modulators (PAMs) of TRPV1 activation. By high throughput screening we discovered several new chemical compounds that enhance the activation of TRPV1 upon orthosteric agonist (capsaicin) binding or by elevated H+ ions. Medicinal chemistry efforts yielded DPM-32 and DPM-74, which are active in in vivo or in vitro assays. These experiments reveal a new approach to pain modulation and pain control and support the idea that TRPV1 agonist activity, induced in several different ways, has the potential to yield novel, non-narcotic, non-addictive, selective, long-lasting analgesic agents that may be effective in acute, persistent, or chronic pain disorders. During this cycle we also published a report on a protein therapeutic agent that is a conjugate between Substance P and a bioengineered Pseudomonas exotoxin. This agent is endocytosed by the substance P receptor expressing second order spinal cord dorsal horn neurons and the exotoxin moiety stops protein synthesis thereby killing the neuron and interrupting the pain pathway to the brain. This is a potent analgesic agent. We intend to test it in certain pain indications, including cancer pain and spinal cord injury pain. Research is in progress to generate high-expressing constructs for SP-PE35 trials. Basic Pain Mechanisms: Underlying the translational and clinical studies are investigations of molecular biology, neuronal function, behavior, and mechanisms of pain transduction. We systematically investigate molecular alterations at the first three steps in the pain pathway beginning with injured peripheral tissue, the dorsal root ganglion and the dorsal (sensory) spinal cord in order to obtain a foundational, comprehensive, quantitative molecular understanding of nociceptive processes related to inflammation and nerve injury. We are using a method called RNA-Seq to sequence all of the mRNAs in a given tissue or cell population. Our work now combines RNA-seq as a component in most of our investigations. We also investigate humans with genetic variations that affect pain sensitivity. At present we are investigating two groups of patients with copy number variants that decrease pain sensitivity. The results are both compelling and informative, and define previously unidentified genetic substrates that can govern pain sensitivity. We also use RNA-seq to define genes involved in inherited peripheral neuropathies and pain channelopathies. These investigations provide new quantitative assessments of neurons of the nociceptive circuit. Through this basic research we aim to obtain a deeper understanding of mechanisms that trigger acute pain and sustain chronic pain and to identify molecular components to control pain. Are any products or services commercially available or being developed that have arisen from the research in this project? Yes. Resiniferatoxin is being developed to treat a variety of different acute and chronic pain conditions. New uses of RTX may form the basis of additional patents. Our RTX patent has been licensed and is now in entering new clinical trials in cancer pain and in osteoarthritis pain. The positive allosteric modulator compounds (PAMs) may also be developed as analgesic agents. Like RTX these would constitute a new class of peripherally acting, non-addictive, non-narcotic therapeutic agents for pain control. However, the field at large does not have a good understanding of endogenous agonists. Understanding endogenous agonists is a necessary prerequisite for therapeutic use of a PAM and more research is required. The substance P-Pseudomonas exotoxin (SP-PE35) is a novel approach to pain control. We are engineering high level expression of the PE35 component for use in clinical trials. New gene discovery is helping to shape our basic knowledge of the molecular composition of pain-sensing neurons and the dynamic changes they can undergo. This knowledge can be used to understand acute and chronic pain disorders.